Amplification using conserved sequence primers

Initial PCR amplification attempts utilised primer pairs F1/R3 and F2/R4. Figure 3.7 shows the agarose gel. The F1/R3 primer pair yielded no visible product (data not shown) but the F2/R4-primed amplifications have generated a single band of size 1200-1300bp, in excellent agreement with the expected size of approximately 1240bp estimated from the murine and bovine cDNA sequences. Since this fragment would be expected to span 11 introns, based upon the known intron/exon mapping of the murine (Pittler and Baehr, 1991),

and the human pdeb genes (Weber et al, 1991), no band would be expected to have been

generated from any contaminating genomic DNA.The identity of the product was confirmed by performing a semi-nested PCR using primer F2 in combination with primer R3.

The purpose of this experiment was twofold: (i) to confirm the nature of the F2/R4 fragment, and (ii) to test the performance of primer R3. The failure of the F1/R3 PCR was tested by using each oligonucleotide in combination with other primers.

Figure 3.8 shows that the presence of F2/R3 product at the expected size both confirms the nature of the template band and proves that primer R3 will work successfully in the dog. FI was tested in combination with the murine primer W118 (which maps to the 3' end of the first exon in mouse and human) on genomic DNA (figure 3.9). A strong band of the expected 450bp size is amplified in both canine and human genomic DNAs whilst Primer FI appears to work equally as well as the murine primer W109 in combination with W118.

Dideoxy chain termination sequencing of the PCR products F1/W118 (section 2.8.3.3) and F2/R4, the latter utilising a biotinylated R4 (R4B) primer with the streptavidin-

C iu in u I c n s d tio n a f p d f h in tuirnutl tln^s

M 1

Figure 3.7 Agarose gel of tlie first pdeb PCR product to be amplified from canine retinal cDNA. Duplicate primer pairs were used on 2pl (lanes 1 and 3) and 1 pi (lanes 2 and 4) aliquots of the cDNA. Lanes 1. 2 primer pair F2/R4, lanes 3 ,4 primer pair F2/R4B (i.e. a biotinylated primer). Lane 5 no DNA control for all 3 primers. M = X/Hindlll markers. Size of bands is given in kilobase pairs.

t

2.0" -

0.5 ►

1 2 3 4 5 6 7

Figure 3.8 Semi-nested PCR of the F2/R4 PCR products using primers F2 and R3 to confirm the nature of the template and to test primer R3. Lanes 1-5 F2/R3 PCRs on 4 aliquots of the F2/R4 product (lane 5 no DNA control), lanes 6 and 7 positive control F2/R4 re-PCR. Size of bands is given in kilobase pairs.

2 3 0 -

2.0-

0 . 5 -

Figure 3.9 Amplification of exon 1 from D B N l.l genomic DNA using primers FI and W 118 (lanes 1-3) and W 109 and W 118 (lanes 4-6). Lanes 3 and 6 contain no DNA control PCRs. M = X/Hindlll markers. Size of bands is given in kilobase pairs.

Characterisation o f p d e b in norm al dogs

magnetic bead methodology (section 2.83.4) was carried out. Following visualisation upon denaturing polyacrylamide gel electrophoresis (section 2.8.4), the nucleotide sequence provided a preliminary confirmation of the nature of the PCR products. They were highly homologous, but not identical to the human, murine and bovine pdeb cDNA sequences in the database. An additional primer, F5 was synthesised, based upon this sequence information and used, in tandem with R4B to generate a nested set of PCR products from the F2/R4 template to test the primer and to facilitate further sequencing. Figure 3.10 shows a series of biotinylated pdeb PCR products which were subsequently used for direct sequencing as detailed in section 2.8.3 4.

The conclusion that the FI binding site on the original cDNA template was not present prompted the decision to extract poly-(A)+ retinal RNA, and to use Superscript RNase H" RT, a cloned M-MLV reverse transcriptase with a deleted RNase H domain, in an attempt to extend the full length of the transcript. The RNA extraction was carried with a Pharmacia

QuikPrep Micro kit (section 2.2.5)

Retinal cDNA from dog DN2 was synthesised using both oligo-dT 1218 and another

primer, R7 which was 100% conserved between the murine, bovine and human (database accession number 841458) pdeb sequences and situated approximately 860bp upstream of the ATG translational start codon. R7 was used to maximise the chances of obtaining the 5' coding region of the transcript and to generate PCR products overlapping those already obtained.

Amplification used the primer pair F1/R7 and the results are shown in figure 3.11. The two aliquots of R7-primed cDNA show a significantly greater amount of the desired PCR product than the oligo-dT-primed cDNA. This may reflect a greater concentration of the template present in the cDNA although PCR under these conditions is not considered to be

quantitative (Gilliland et al, 1990; Wang and Mark, 1990). If product yield is related to

template, then far more of the desired template was reverse transcribed from the transcript- specific primer R7 than from the oligo-dT. The proximity of the R7 primer to the 5' end of the coding region may have increased the amount of fully extended cDNA relative to that

( 'h ti h u I c n s tilio n t>f /n lc h in ru n n u il d o ^ s

M 1

Figure 3.10 PCR pr(xlucts generated using biotinylated primer R4B with F2 (lanes 6-9) and semi nested from this template with primer F5 (lanes 1-4). These products were used for direct sequencing, lane 5 contains the no DNA control PCR. M = W in d lll markers.

0 . 5 K B ►

4 5 IVI 6 7 8

Figure 3.11 PCR product generated from cDN A using primers FI and R7. Equal amounts of RNA from dog DN2 were used in the cDNA synthesis and PCR but for lanes 1 and 3, the first strand synthesis was primed with R7 itself, whilst for lane 2. oligo-dTi2-i8 was used. Lane 4 is the no DNA control PCR The expected band size of about 860 bp was obtained. M = A,/PvuII markers. Sizes of bands are given in base pairs.

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8 6 0 " -

Figure 3.12a F1/R3 PCR product obtained from retinal cDNA at the expected size of about 1680 bp (lane 1). Lane 2 shows the no DNA control reaction. M = ^ /l^ u ll markers.

Figure 3.12b W80/R3 (lane 3) and W80/W127 (lane 4) PCR products obtained from retinal cDNA showing the expected sizes. Lane 1 contains the no DNA control PCR. M = A/Hindlll markers. Band sizes shown in kilobase pairs.

1 7 0 0

C haracterisation o f p d e b in norm al dogs

extended from the poly-(A) tail, despite the ability of the Superscript enzyme to extend for much longer distances than approximately 3kb.

To facilitate sequencing of PCR fragments and to generate additional cDNAs

overlapping those already obtained, the oligo-dTi2-i8 ptimGd cDNA was used as a template

to PCR-amplify F1/R3, W80/R3 and W80/W127 fragments. The latter two are semi- and fully- nested at either end relative to the former and the primers are derived from murine nucleotide sequence. The results are shown in figure 3.12a and 3.12b. Single fragments of the expected size were obtained from the cDNA in each case.